Diabetes is a metabolic disease occurring in people with high blood sugar, and can lead to serious damage to many systems of the body, especially the nerves and blood vessels. According to the expectation of the World Health Organization, the number of people with diabetes in China has been ranked first in the world. Diabetes will be the most serious public health problem in China in the next 50 years.
Glycated albumin (GA) is the product formed by a non-enzymatic reaction occurring between glucose and glycated N-terminus albumin in human serum. 90% of them reacts with albumin within the chain of lysine ε-NH2 residue. The reaction principle lies in, at first both of them form an unstable Glycosylamine or Schiff Base. Then, through irreversible Amadori rearrangement, the latter forms a stable amino ketone (ketone amine). Since the half-life of albumin is about 20 days, detection of glycated albumin can be used to detect the average level of blood sugar over the past 2-3 weeks. Currently, glycated albumin testing has become an essential test for diabetics. Compared with glycated hemoglobin, it is more suitable as an index for assessing the risk of hospitalization and death of diabetic patients who have had dialysis.
Therefore, how to accurately determine the amount of glycated albumin in human's serum has become the key of clinical detection of glycated albumin. Currently, the market mainly uses enzymatic method to detect glycated albumin in human's serum. The reaction principle lies in, using protease at first, glycated albumin is digested into glycated polypeptides with a low molecular weight. Then, using fructosyl amino acid oxidase to catalyze glycated polypeptides to perform the oxidation reaction to generate polypeptides (or amino acids), Arabino hexose and H2O2. Release of H2O2 is detected by an endpoint reaction colorimetric method. Absorbance at 600 nm is proportional to the concentration of glycated albumin. Specific reaction process is as follows:
As is known form the above reaction mechanism and reaction steps, fructosyl amino acid oxidase is the key enzyme of detecting glycated albumin, and fructosyl amino acid oxidase has become the key for whether glycated albumin in human's serum can be accurately detected.
Fructosyl amino acid oxidase can also be referred as Fructosyl amino acid enzyme. Amadoriase, ketone oxidase, and etc. Such fructosyl amino acid oxidase has already been found in many bacteria, yeasts, and fungi, for example, from Aspergillus, Penicillium, Fusarium, Pichia, Coniochaeta, Eupencillum, Corynebacterium, and etc. (non-patent document 1, Lin, Z. and J. Zheng (2010) “Occurrence, characteristics, and applications of fructosyl amine oxidases (amadoriases).” Appl Microbiol Biotechnol 86. (6): 1613-1619). The above fructosyl amino acid oxidases are all possible to be applied in the glycated albumin test kit. However, at the same time, since in practice the diagnostic test kit need to be stored for a certain period of time, the requirements for stability are relatively high. Thus, the raw material-enzymes used also need to be stable. However, the thermostability of the enzymes shown in the above public materials are not ideal. After a heating treatment at 45° C. for 10 minutes, fructosyl amino acid oxidase from Aspergillus terreus GP1 shows a residual enzyme activity of about 40% (Non-Patent Document 2: Yoshida, N., Y. Sakai, et al (1996) “. Primary structures of fungal fructosyl amino acid oxidases and their application to the measurement of glycated proteins” European Journal of Biochemistry 242 (3): 499-505). After a heating treatment at 45° C. for 5 minutes, fructosyl amino acid oxidase from Fusarium oxysporum S-1F4 shows a residual enzyme activity of about 10% (Non-Patent Document 3: Sakai, Y., N. Yoshida, et al (1995). “Purification and properties of fructosyl lysine oxidase from Fusarium oxysporum S-1F4” Bioscience, biotechnology, and biochemistry 59 (3): 487). After a heating treatment at 37° C. for 30 minutes, fructosyl amino acid oxidase from the Coniochaetidium savoryi ATCC36547 shows a residual enzyme activity of 80% (Japanese Patent Application Publication No. 2004-275013;). Because of the poor thermostabilities of existing fructosyl amino acid oxidases, the stability of glycated albumin test kits on the market nowadays cannot meet the requirements. In many cases, a large amount of stabilizer is added in the test kit, so as to increase the stability of the test kit (International Patent Application Publication No. WO02002061119A1). This leads to a higher viscosity of the reagent, as well as a series of other issues. It is not conducive to be applied in a biochemical analyzer.
Therefore, there is an urgent need to develop a fructosyl amino acid oxidase with high thermostability. Also, this fructosyl amino acid oxidase is used to develop a glycated albumin test kit having both high sensitivity and long-term stability.